University of Groningen Multiple aspects of a plasma cell dyscrasia de Waal, Elisabeth Geertruida Maria

(1)

Multiple aspects of a plasma cell dyscrasia

de Waal, Elisabeth Geertruida Maria

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date: 2018

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

de Waal, E. G. M. (2018). Multiple aspects of a plasma cell dyscrasia. Rijksuniversiteit Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

Tyrosine

Methionine

LAT1

protein

synthesis

glucose

pathway

fatty acid

amino acid

sterols

G LUT

_VEGF

acetate

TC+

mitochondria

golgi

nucleus

Ac-Coa

Hypoxia

nitroimidazole

Oxygen

radical

FLT

CXCR4

VLA

VCAM

TK

stromal cell

Choline

CD38

CD138

CHAPTER 4

[18F]-FDG-PET increased visibility of bone lesions in

relapsed multiple myeloma: Is this hypoxia driven?

Esther G.M. de Waal1

Riemer H.J.A. Slart2

Marnix J. Leene1

Philip M. Kluin MD3

Edo Vellenga1

Departments of Hematology1_{, Nuclear Medicine and Molecular Imaging}2_,

and Pathology3_{University Medical Center Groningen, Groningen, The Netherlands.}

(3)

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39

Abstract

Introduction: Whole body X-ray (WBX) is used for detecting skeleton abnormalities in multiple myeloma (MM) patients. An alternative might be [F-18]fluorodeoxyglucose positron emission tomography (FDG-PET) which make use of metabolic changes of malignant plasma cells. The aim of this study is to demonstrate whether [18F]-FDG-PET is more valuable in relapsing MM compared to WBX and to define its prognostic value. In addition 1-α-D: -(5-deoxy-5-[18F]-fluoroarabinofuranosyl)-2-nitroimidazole ([18F]-FAZA) scan and immunohistochemical staining on bone marrow was performed to define whether FDG uptake coincides angiogenesis related tumor hypoxia.

Methods: [18F]-FDG-PET (n=44) and [18F]-FAZA PET (n=5) was performed in patients with relapsed MM. Bone marrow biopsies (n=20) were evaluated for hypoxia inducible factors (HIF) -1α and -2α, vascular endothelial growth factor (VEGF), glucose transport protein (GLUT) 1 and 3 and the micro-vessel density (MVD).

Results: New lesions were more frequently demonstrated on [18F]-FDG-PET than on WBX (p=0.000001). [18F]-FDG-PET was not predictive for progression free survival and overall survival. Immunohistochemical staining on bone marrow biopsies demonstrated a significant increase in MVD and elevated expression of VEGF, HIF-2α and GLUT-3 by the malignant plasma cells. However HIF-1α expression and [18F]-FAZA scans were negative.

Conclusion: Our results demonstrate that [18F]-FDG-PET is relevant for diagnostic purposes compared to WBX in relapsing MM. The enhanced uptake of [18F]-FDG-PET is likely related to the activation of the HIF-2α signaling pathway, but probably independent of hypoxia induced signaling in view of the negative findings on both [18F]-FAZA PET and HIF-1α expression.

Keywords: relapsing multiple myeloma, [18F]-FDG-PET, [18F]-FAZA, immunohistochemical

(4)

4 Introduction

Multiple myeloma (MM) is a disease characterized by a monoclonal plasma cell population in bone marrow whereby osseous involvement is a predominant feature. In 90% of the patients’ lytic bone lesions develop, which is an important cause of morbidity1_{. Lytic bone lesions are}

the result of increased bone resorption and reduced bone formation2_{. Whole body X-ray}

(WBX) is the method of choice for detecting skeleton abnormalities. This technique has several limitations such that it can detect only lesions that have lost more than 30% of the trabecular bone3_{. Furthermore the value in relapsing disease is limited because lesions persist after}

treatment so it cannot distinguish old vs. new active skeleton lesions. Therefore alternative techniques have been developed to visualize disease activity. 18-F-fluorodeoxyglucose positron emission tomography ([18F]-FDG-PET) uses the enhanced metabolic activity of cells to visualize the abnormal lesions4_{. In newly diagnosed MM patient [18F]-FDG-PET detects}

more lesions compared to WBX; furthermore, a positive [18F]-FDG-PET scan is of prognostic value5_{. This imaging technique may also be useful in patients with relapsing MM since it is not}

hampered by the presence of preexisting skeletal defects and visualizes areas of enhanced metabolic activity6-8_{. Indeed, in our previous study we demonstrated that [18F]-FDG-PET is}

a valuable tool for detecting MM activity in patients with relapsing MM9_{. In view of these}

encouraging results we extended the number of included patients having a [18F]-FDG-PET and a skeletal survey in the work up of relapsing MM to evaluate whether [18F]-FDG-PET indeed detects more lesions in patients with relapsing MM and has a prognostic value in the relapse setting of MM as well.

The enhanced metabolic activity defined by [18F]-FDG-PET could be related to hypoxia and the subsequent increased protein expression of hypoxia inducible factors (HIF) 1α and 2α, which can trigger the vascular endothelial growth factor (VEGF) pathway and stimulate angiogenesis10,11_{. Previous studies have shown that angiogenesis defined by increased}

micro-vessel density (MVD) is highly correlated with progressive disease in MM12_{. Recently new}

scanning methods have been developed for demonstrating in vivo tumor hypoxia. The PET tracer hypoxia, 1-α-D:-(5-deoxy-5-[18F]-fluoroarabinofuranosyl)-2-nitroimidazole ([18F]-FAZA), has been shown to accumulate in tumor hypoxia. In a study performed by Postema

et al., 50 patients with different types of malignancy (solid as well as hematologic), tumor

hypoxia could be demonstrated in 46% of the patients13_.

Based on these findings we studied in vitro hypoxia related proteins including HIF 1α and 2α, VEGF and other hypoxia related proteins such as glucose transporter (GLUT) 1 and -3 expression on bone marrow biopsies of MM patients and determined MVD. In addition, in

vivo [18F]-FAZA scans were performed in these patients to demonstrate whether tumor

hypoxia can also be visualized in vivo in MM patients and whether this corresponds with elevated uptake of [18F]-FDG-PET.

(5)

Materials and Methods

Patients

A total of 44 MM patients were prospectively included in this study. Approval for the study was obtained from the local medical ethical committee. The diagnosis MM was based on the presence of a monoclonal plasma cell population in the bone marrow, the presence of a monoclonal immunoglobulin protein or elevation of free light chain (FLC) in serum (normal range FLC kappa 2.3 – 20.0 mg/l and FLC lambda 4.4 – 32.0 mg/l) and the presence of one or more of the following related organ or tissue impairments; lytic bone lesion, renal involvement, hypercalcemia or anemia. Relapse MM after having achieved CR was defined as follows: 1. reappearance of paraprotein; 2. more than 5% plasma cells in bone marrow; 3. new lytic bone lesions or progression of old lesions; 4. new hypercalcemia. Relapse MM after having achieved PR was defined as follows: 1. increase of paraprotein with more than 25%; 2. increase of urine paraprotein with more than 25%; 3. increase of plasma cells in bone marrow with 10%; 4. new lytic bone lesions or progression of old lesions; 5. new hypercalcemia, according to international guidelines14_.

In all patients with relapsing MM in our institute, a [18F]-FDG-PET scan was performed. A skeletal survey examination was performed in 38 patients. A subgroup of patients with a positive [18F]-FDG-PET scan result were randomly chosen to undergo an [18F]-FAZA. Patients were treated according to ongoing trials or protocols15-17_{. Of 44 patients, 37 (75%)}

were previously treated with autologous stem cell transplantation. Most of the patients were treated upfront with three cycles of induction with VAD (vincristine, doxorubicin and dexamethasone), TAD (thalidomide, doxorubicin and dexamethasone) or PAD (bortezomib, adriamycin and dexamethasone). This was followed by peripheral blood stem cell collection and transplantation. Treatment schedules used at relapse included bortezomib, lenalidomide, thalidomide, dexamethasone and in some cases in combination with cyclophosphamide or melphalan. At last follow up 26 of the 44 patients are alive.

Response to treatment was measured using the standard criteria, although not all patients underwent a repeated bone marrow biopsy to establish complete response. CR is defined as no m-protein or FLC present and normal bone marrow, very good partial response (VGPR) is defined as more than 90% reduction in m-protein or FLC. PR is defined as a more than 50% reduction in m-protein or FLC and progressive disease as an increase in m-protein or FLC with more than 25%14_.

Immunohistochemistry for MVD, VEGF-A, HIF 1α and 2α and GLUT-1 and -3

The expression of angiogenesis and hypoxia related features was examined by immunohistochemical staining on bone marrow biopsies (n=20). After fixation in 10% neutral phosphate buffered formalin (3.6% formaldehyde) for at least 12h, decalcification in a solution

(6)

4

containing 10% (v/v) acetic acid and 10% formalin (v/v; 3.6% formaldehyde) for 1 or 2 days. Than the bone marrow was paraffin embedded and cut in sections of 4 µm and stained as follows. Slides were dewaxed and rehydrated in graded alcohol solutions. Endogenous peroxidase activity was blocked with hydrogen peroxide. Antigen retrieval was done heat-induced with the microwave, expect for the VEGF staining were antigen retrieval was done with protease 0.1%. Slides were than incubated with the primary antibody for GLUT-1 (rabbit polyclonal 07-1401, Millipore USA; diluted 1:750), GLUT-3 (rabbit polyclonal ab15311, Abcam UK; diluted 1:100), HIF-1α (mouse monoclonal clone 54/HIF-1α BD Biosciences; diluted 1:70) HIF-2α (mouse monoclonal ab8365, Abcam; diluted 1:200) and VEGF-A (rabbit polyclonal sc-152, Santa Cruz USA; diluted 1:50). Blood vessels were visualized with a CD34+ antibody (mouse monoclonal QBEnd 10, Dako, Copenhagen, Denmark). The slides were washed with PBS and after that incubated with a rabbit anti mouse or goat anti rabbit horse radish peroxidase conjugated as secondary or tertiary antibody. For the VEGF staining streptavidine peroxidase was used as a tertiary antibody. The chromogenic reaction was performed with diaminobenzidine for 12 min and after that sections were counterstained with haematoxylin. Finally, the slides were dehydrated and mounted.

The intensity of staining was analyzed semi-quantitatively. The percentage of positive plasma cells was counted according the following scoring system: no visibility, 10-30% positive, 30-50% positive, 50-80% positive and more than 80% positive plasma cells18_{. The vessel count}

was measured using light microscopy in areas of the slide containing highest numbers of blood vessels per selected area (hotspot). After the hotspots were identified, the total number of vessels per selected image was counted at 400 magnifications. At least five fields were counted for each section and the true vessel number was expressed as the mean of 5 counts19_.

Skeletal survey

Each patient underwent a whole body X-ray survey, consisting of X-skull, X-humeri, X-femora, X-whole spine, X-pelvis. Conventional radiographs were acquired at 73 kVp and 16 mAs by using a radiographic imager (MULTIX Swing; Siemens Medical Systems, Chicago, Ill) by using a bucky grid.

[18F]-FDG-PET

All patients had to fast for at least 5 h before undergoing [18F]-FDG-PET. FDG was administered intravenously (4-5 MBq/kg). After an uptake period of 60 min, PET emission data were acquired from total body, 5 min per bed position. A Biograph mCT scanner was used (Siemens Medical Systems, Knoxville, TN, USA). The measured resolution is 2-4 mm in full width at half maximum transaxially in the center of the field of view. For PET data reconstruction 3 interactions, 21 subsets, with an image size of 256 x 256, zoom 1, were used.

(7)

FDG uptake was quantified using the maximal standardized uptake value (SUVmax) analysis in all patients. A standardized protocol for quantification of SUVmax was used, including blood glucose correction20_{. The region of interests used in SUV analysis were 3-dimensional}

and were based on the max value within the 50% isocontour boundaries using a Siemens workstation (Siemens Medical Systems, Knoxville, TN, USA).

The number, size, localization and SUVmax of focal lesions were recorded. The [18F]-FDG-PET scan was either negative with no focal lesions or positive with focal lesions. In addition, definition by Zamagni et al was used, defining PET positivity as more than 3 lesions or SUVmax > 4.25_.

The skeletal survey and [18F]-FDG-PET were performed within a month of each other. [18F]-FDG-PET images were examined by an experienced nuclear physician using also visual examination with special attention to the skeleton.

[18F]-FAZA

Procedures for good-manufacturing-practice production of the hypoxia tracer [18F]-FAZA have been developed previously. The synthesis of [18F]-FAZA was optimized using a Micro Fluid Chemistry Module (Advion). The routine production was performed using a robot system (Zymark). Briefly, the precursor (2 nitro imidazole) for labeling [18F]-FAZA was reacted with dried 18F/K222 complex in dimethyl sulfoxide and thereafter deprotected with 0.1 M NaOH. After high-performance liquid chromatography purification of the reaction mixture,

[18F]-FAZA was formulated using an Oasis HLB plus cartridge. The final sterile solution was

analyzed using high-performance liquid chromatography and released for administration to the patient.

[18F]-FAZA PET scans were performed on the same mCT camera as the [18F]-FDG images according to local standard operating procedures for [18F]-FAZA PET scans. Patients were injected with 370 MBq intravenously. After a waiting period of 120 min, a scan was obtained from the midthigh to the brain and analyzed using the previous-mentioned research workstation. [18F]-FAZA SUVmax was quantified in the same way as [18F]-FDG SUVmax, including correction for the partial-volume effect. The median time interval between [18F]-FDG PET and [18F]-FAZA PET was 21 days (range 1-51). The data were reconstructed with time-of-flight, high-definition, ordered-subsets expectation maximization using 3 iterations, 21 subsets, and a 5-mm gaussian postprocessing filter and had a spatial resolution of 2.04 × 2.04 × 2 mm.

Statistical analysis

Difference between the amount of lesions between WBX and [18F]-FDG-PET were calculated from the data using a Wilcoxon signed ranks test. Survival was calculated using the method of Kaplan and Meier and compared by log rank test. Correlation was calculated using a Spearmans rho. P < 0.05 was considered significant.

(8)

4 Results

This study included 44 patients. The group consisted of 27 men and 17 women with a median age of 62.5 years (range 48 to 78 years). Median number of prior treatment regimens was 3.5 (range 2-12). Patient characteristics are presented in table 1. Fifty-five percent of patients showed production of monoclonal IgG, 8% of IgA and 35% patients only showed production of serum FLC.

Table 1: Patient characteristics

Characteristic (n = 44) Number % Age (years) median 62 range 48 – 78 gender male 27 61 female 17 39

no of prior treatment regimens

median 3.5 range 02 – 12 Type of m-protein IgG 27 55 IgA 4 8 IgM 0 0 FLC only 17 35

% plasma cells bone marrow at relapse MM

No bone marrow 4 10

1-Oct 22 45

Oct-30 17 35

> 30 5 10

Type of prior treatment

Autologous stem cell transplantation 42 86

Allogeneic stem cell transplantation 6 12

Lenalidomide 32 65

Bortezomib 43 78

Thalidomide 38 76

Low dose melphalan 14 29

Legend: FLC; free light chain. MM; multiple myeloma.

During the follow up 5 patients experienced two episodes of relapse. For each relapse [18F]-FDG-PET was performed. Not in all cases a skeletal survey was performed in combination with [18F]-FDG-PET. In total 49 [18F]-FDG-PET scans were performed and 38 skeletal survey examinations. In 79% of the patients pre-existing defects were demonstrated on the skeletal survey. New abnormalities at relapse were demonstrated in 42% of the patients. The median

(9)

number of skeletal lesions on X-ray was 0 (range 0-7). The [18F]-FDG-PET showed abnormal uptake in 82% of the patients. No diffuse increased FDG uptake in bone marrow was noticed in the studied patients. The median number of FDG lesions was 4 (range 0-22). The average SUVmax was 5.4 with a highest SUVmax of 14.1 and a lowest SUVmax of 1.7. The number of lesions was significantly higher with the [18F]-FDG-PET vs. skeletal survey (p = 0.000001). One patient had a lesion in the skull, not detected on [18F]-FDG-PET. One other patient had several small lesions (1 cm or smaller) in pelvic and femur also not detected with [18F]-FDG-PET. None of the patients received treatment before the skeletal survey and [18F]-FDG-[18F]-FDG-PET. All other lesions defined by the skeletal survey were confirmed on the [18F]-FDG-PET. In addition, in 5 patients extra-osseous lesions were detected with strong positive FDG avid lesions (median SUVmax 9.7). None of these lesions were demonstrated on plain X-ray. For 9 patients no abnormal lesions could be demonstrated on both [18F]-FDG-PET and WBX although all patients had distinct signs of relapsing disease.

Treatment results were available for all 44 patients. CR was obtained in 13 (27%) of the patients, VGPR in 6 (12%) patients, PR in 13 (27%) patients and progressive disease in 13 (27%) patients. For 3 patients it was not possible to measure treatment response, 1 patient was lost during follow up, 1 patient died due to another cause and for 1 patient the follow up interval was too short. Median follow-up was 14 months (range 2-49). Median progression free survival (PFS) was 11.7 months (range 2-43). For patients reaching a CR the median PFS was 16 months compared to 5.3 months for patients with progressive disease (p= 0.00001). Overall survival was not reached for patients with CR vs. 8 months for patients with progressive disease (p = 0.000001).

In 12 patients [18F]-FDG-PET was performed after chemotherapy treatment. Nine patients had responsive disease with decrease in the number of lesions according to the [18F]-FDG-PET. Of 9 patients, 3 (33%) had a CR on the [18F]-FDG-[18F]-FDG-PET. Two patients had stable disease and 1 patient had progressive disease. On the [18F]-FDG-PET before treatment there were median 7.5 lesion (range 1-22) and after treatment 3 lesions (range 0-10). The median SUV max declined from 5.5 (range 3.3-9.4) to 2.27 (range 0-4.6) (p=0.003). Response observed with [18F]-FDG-PET was comparable to clinical response.

Overall, [18F]-FDG-PET findings were not predictive for PFS (p =0.30) and OS (p=0.33) and also when PET positivity was defined as more than 3 lesions or SUVmax > 4.25_{. Figure 1A and}

(10)

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 57

4

Figure 1A: Progression free survival correlated to FDG-PET outcome

1

p= 0.33

FDG-PET neg --- FDG-PET pos

Legend: The correlation between [18F]-_{[18F]-FDG-PET outcome and progression free survival.}

Figure 1B: Overall survival correlated to FDG-PET outcome

p= 0.30

FDG-PET neg --- FDG-PET pos

(11)

Immunohistochemical staining on bone marrow biopsies (n=20) with the CD34 monoclonal antibody demonstrated an increased MVD scoring of median 13.5 (range 3-200) vs. 3.5 on normal bone marrow21_{. The MVD was particular high when clusters of plasma cells were}

present. In 80% of the bone marrow biopsies more than 50% of the plasma cells were positive for VEGF-A. The staining for the GLUT demonstrated that 10% of the biopsies showed more than 50% of the plasma cells positive for GLUT-1 and in 55% of the biopsies for GLUT-3. A clear distinction was observed for HIF-1α and HIF-2α; no staining for HIF-1α was demonstrated on plasma cells although the surrounding endothelial cells demonstrated a distinct positive staining, while in 85% of the samples more than 50% of the plasma cells were positive for HIF-2α. Table 2 shows the results of immunohistochemistry combined with the results of the [18F]-FDG-PET and treatment outcome. Figure 2 provides the results of the staining from 1 patient. MVD was the only factor that correlated with [18F]-FDG-PET positivity (p = 0.03). The expression of VEGF (p = 0.67), HIF-2α (p=0.33) or GLUT-3 (p =0.50) was not predictive for having a positive or negative [18F]-FDG-PET result.

(12)

4

Table 2: Findings of immunohis tochemic al s taining , [18F]-FDG-PET and r esponse t o tr ea tmen t Nr VE GF MVD GL UT -1 GL UT -3 HIF-1 α HIF-2 α FDG-PET FDG-PET > 3 SUV -MAX Respons t o tr ea tmen t 1 >80% 5 50-80% 10-30% neg 50-80% pos pos 14,5 pr ogr disease 2 >80% 3 neg >80% neg >80% neg neg 0 CR 3 >80% 3 neg neg neg 50-80% pos pos 13,2 PR 4 >80% 14 neg neg neg 50-80% pos neg 3,7 CR 5 >80% 85 neg neg neg 30-50% pos pos 6,4 PR 6 30-50% 4 neg neg neg 10-30% pos pos 4,5 CR 7 >80% 200 10-30% >80% neg >80% pos pos 4,3 CR 8 50-80% 4 50-80% 999 neg 50-80% neg neg 0 -9 30-50% 13 neg 50-80% neg >80% pos pos 8 PR 10 >80% 6 10-30% 50-80% neg >80% neg neg 0 pr ogr disease 11 50-80% 23 neg neg neg >80% pos pos 8,4 pr ogr disease 12 50-80% 12 neg 50-80% neg >80% pos pos 22,2 pr ogr disease 13 >80% 155 neg >80% neg >80% pos pos 3,2 PR 14 >80% 86 30-50% >80% neg >80% pos neg 4,6 PR 15 >80% 26 10-30% >80% neg 50-80% pos pos 5,6 VGPR 16 >80% 72 10-30% >80% neg >80% pos pos 22,2 pr ogr disease 17 10-30% 25 neg 10-30% neg 50-80% pos neg 5,3 PR 18 10-30% 4 neg 10-30% neg 30-50% pos neg 12,3 † 19 >80% 40 neg >80% neg >80% pos pos 6,5 VGPR 20 50-80% 5 neg 50-80% neg >80% neg neg 0 SD Leg end: The findin gs of immunohis tochemic al st aining , FDG-PET and response to tr ea tmen t. GL UT ; gluc ose tr ansport er , HIF; hypo xia inducible fact or s, VE GF; vascular endothelial gr owth fact or , MVD; micr o-vessel density , FDG-PET>3: mor e than 3 lesions found on the FDG-PET as de fined by Zama gni [5]. SUV -ma x; ma ximal st andar diz ed up tak e value, CR; comple te response, VGPR; ver y good partial response, PR; partial response, SD; st able disease , pr ogr disease; pr ogr essiv

e disease. † Died of other c

ause. P atien t number 8 los t t o f ollo w up.

(13)

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 60

Figure 2: Immunohistochemical staining for GLUT-1 and 3, HIF-1α and HIF-2α, VEGF and MVD

Legend: Immunohistochemical staining for A:GLUT-1, B:GLUT-3, C:HIF-1α, D:HIF-2α, E:VEGF and F:MVD. GLUT; glucose transporter, HIF; hypoxia inducible factors, VEGF; vascular endothelial growth factor, MVD; micro-vessel density.

Five randomly chosen patients (4 male and 1 female) underwent an [18F]-FAZA PET scan. All five patients had a positive FDG-PET scan. They received no treatment before the [18F]-FAZA scan. They had a median of 0 (range 0-1) lesions on the WBX and a median of 7 (range 2-15) lesions on the [18F]-FDG-PET. The median SUVmax was 9.4 (range 4.2-13.6). No lesions were demonstrated on [18F]-FAZA PET. Figure 3 demonstrates a patient with clear [18F]-FDG-PET positivity of a large extramedullary lesion but without uptake on [18F]-FAZA [18F]-FDG-PET.

1 A B C D E F A B C D E F A B C D E F 1 A B C D E F A B C D E F A B C D E F

(14)

4

Figure 3: [18F]-FDG-PET and [18F]-FAZA of the same patient with relapsing MM

1

A B

Legend: [18F]-FDG-PET and [18F]-FAZA image of patient (number 16 in table 2) with progression of MM. Large extramedullary and multiple ossal lesions are visible on the [18F]-FDG-PET scan (left scan). No lesions were visible on the [18F]-FAZA scan (right scan).

Discussion

Skeletal survey is the standard method for the detection of skeletal lesions in patients with MM. In relapsing disease measurement of disease progression is difficult to assess with this method because of the presence of pre-existing skeleton abnormalities. New techniques are therefore explored to demonstrate disease progression. In particular, MRI scanning is recommended for detecting osteolytic lesions in the spinal and pelvic regions. The disadvantage of this technique is the difficulty to use it for response evaluation since it takes 9-12 months before lesions resolve on MRI8_{. In addition, it has limitations for detecting bone}

marrow infiltration in ribs, clavicle and skull3_.

In the present study we used the property of enhanced metabolic activity shown by increased glucose uptake by the malignant plasma cells visualized on [18F]-FDG-PET. Thus far studies with [18F]-FDG-PET have been performed in untreated MM patients, demonstrating more positive lesions on the [18F]-FDG-PET scan in comparison to WBX3_{. The present study in}

(15)

patients with relapsing MM also demonstrated a higher number of lesions with FDG-PET as compared with WBX. This is probably due to the fact that over 30% of the trabecular bone must be lost before osteolytic lesions are visible on WBX3_.

In general the lesions found on WBX were also demonstrated with [18F]-FDG-PET, except for small, diffuse defects. It has been demonstrated that small lesions, < 1 cm, are difficult to detect with [18F]-FDG-PET23_{, due to limited resolution of the PET camera. We observed}

new skeletal survey lesions in 42% of the patients. This number might overestimate the real number of skeletal lesions after the last relapse because a number of patients had several relapses and skeletal survey was not performed on every occasion. On the other hand, extramedullary MM will be missed on WBX and can be appropriately diagnosed with [18F]-FDG-PET4_.

[18F]-FDG-PET has become an established imaging modality for the detection of osseous lesions in newly diagnosed MM patients and seems to be an independent prognostic parameter for overall survival5_{. In relapsing MM patients only limited studies have been}

performed. Derlin et al, demonstrated in relapsing MM patients following allogeneic stem cell transplantation a positive [18F]-FDG-PET scan result in 80% of the patients24_{, which}

is comparable to our patients following autologous stem cell transplantation. Overall, we noticed that [18F]-FDG-PET provides more information than does WBX for detecting skeleton lesions in relapsing MM patients.

[18F]-FDG-PET has prognostic value for the treatment results in newly diagnosed MM patients5_{. However, in this cohort of relapsing MM we could not demonstrate the prognostic}

significance for [18F]-FDG-PET positivity. This might be due to the great variability in the number of relapses and prior treatment regimens. Median PFS was 11.7 months (range 2-43), which is comparable to other studies in patients with relapsing disease25_{. The most}

important factor was the response to treatment, patients reaching CR having a better PFS and OS (p=0.00001).

To study in greater detail the cause of the enhanced uptake of [18F]-FDG-PET in MM, we studied different parameters that are linked and may influence FDG uptake activity, such as tumor hypoxia. The PET tracer [18F]-FAZA makes use of this condition. Two-nitroimidazole compounds undergo reduction under hypoxic conditions, forming highly reactive oxygen radicals. This binds to macro-molecules inside the cell. When labeled with a radioisotope, this can be visualized with a PET-scanner. [18F]-FAZA has been used in patients with solid tumor like head and neck cancers and non-small cell lung cancers to detect tumor hypoxia26, 27_.

[18F]-FAZA scan was positive in two third of the head and neck cancer patients and in all non-small cell lung cancer patients26, 27_{. In our study we evaluated the regional uptake of [18F]-FAZA in}

patients with relapsing MM with a positive [18F]-FDG-PET scan result. However, in none of the 5 patients an enhanced uptake of the [18F]-FAZA could be demonstrated.

(16)

4

It is well known that bone marrow of MM patients reveal higher MVD and increased VEGF expression12_{. The expression of VEGF can be regulated by hypoxia inducible factors like}

HIF-1α and HIF-2α. In contrast to the study of Giatromanolaki et al11_{who demonstrated that}

33% of the MM patients stained positive for HIF-1α, no HIF-1α staining of plasma cells was demonstrated in our patients. In addition we observed a low GLUT-1 expression, which is in line with the results of the HIF-1α staining since GLUT-1 is a downstream target of HIF-1α. The negative [18F]-FAZA scan result corresponds also with these findings. However HIF-2α expression was demonstrated in a high number of samples in conjunction with an increase in MVD and elevated expression of VEGF, suggesting that the HIF signaling pathway was activated. The number of MVD also correlated with the positivity on [18F]-FDG-PET. In solid tumor different expression patterns of HIF-1α and HIF-2α are described, suggesting different roles for both factors. It has been proposed that HIF-1α increases directly in response to hypoxia, while HIF-2α is responsive during prolonged period of hypoxia28_{. However, expression}

of HIF or VEGF is not synonymous with tumor hypoxia. Alternative pathways can be activated due to oncogene activation or mutations that trigger the expression of HIF. For example, it has been shown that signal transducer and activator of transcription 5 (STAT-5) can activate the expression of HIF-2α. STAT-5 can be activated by several cytokines and it induces expression of several target genes, including HIF-2α29_{. Whether STAT-5 plays an important role in multiple}

myeloma is not extensively studied but this could explain the observed difference in uptake between [18F]-FDG-PET and [18F]-FAZA.

In summary, our results demonstrate that in relapsing MM patients [18F]-FDG-PET is highly relevant for diagnostic purposes compared with WBX, but has limited value as prognostic parameter. The enhanced uptake of [18F]-FDG-PET is likely related to the activation of the HIF signaling pathway but not related to hypoxia in view of the negative findings on [18F]-FAZA PET.

(17)

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References

1. Rajkumar SV. Multiple myeloma: 2013 update on diagnosis, risk-stratification, and management. Am J Hematol. 2013;88:226-35.

2. Terpos E, Dimopoulos MA. Myeloma bone disease: pathophysiology and management. Ann Oncol. 2005;16:1223-31.

3. Regelink JC, Minnema MC, Terpos E, Kamphuis MH, Raijmaker PG, Pieters–van de Bos IC, et al. Comparison of modern and conventional imaging techniques in establishing multiple myeloma-related bone disease: a systematic review. Br J Haematol. 2013;162:50-61.

4. Agool A, Glaudemans AW, Boersma HH, Dierckx RA, Vellenga E, Slart RH. Radionuclide imaging of bone marrow disorders. Eur J Nucl Med Mol Imaging. 2011 Jan;38:166-78.

5. Zamagni E, Patriarca F, Nanni C, Zannetti B, Englaro E, Pezzi A, et al. Prognostic relevance of 18-F FDG PET/CT in newly diagnosed multiple myeloma patients treated with up-front autologous transplantation. Blood. 2011;118:5989-95.

6. Bredella MA, Steinbach L, Caputo G, Segall G, Hawkins R. Value of FDG-PET in the assessment of patients with multiple myeloma. Am J Roentgenol. 2005;184:1199-1204.

7. Lütje S, Rooy de JW, Croockewit S, Koedam E, Oyen JW, Raymakers RA. Role of radiography, MRI and FDG-PET/CT in diagnosing, staging and therapeutical evaluation of patients with multiple myeloma. Ann Hematol. 2009;88:1161-8.

8. Bartel TB, Haessler J, Brown TL, Shaughnessy JD jr, van Rhee F, Aniasse E, et al. 18-F-fluorodeoxyglucose positron emission tomography in the context of other imaging techniques and prognostic factors in multiple myeloma. Blood. 2009;114:2068-76.

9. De Waal EGM, Slart RHJA, Vellenga E. Is FDG-PET a better imaging tool than somatostatin receptor scintygraphy in patients with relapsing multiple myeloma? Clin Nucl Med. 2012;37:939-42. 10. Vacca A, Ribatti D. Bone marrow angiogenesis in multiple myeloma. Leukemia. 2006;20:193-9. 11. Giatromanolaki A, Bai M, Margaritis D, Bourantas KL, Koukourakis MI, Sivridis E, et al. Hypoxia and

activated VEGF/receptor pathway in multiple myeloma. Anticancer Res. 2010;30:2831-6. 12. Rajkumar SV, Mesa RA, Fonseca R, Schroeder G, Plevak MF, Dispenzieri A, et al. Bone marrow

angiogenesis in 400 patients with monoclonal gammapathy of undetermined significance, multiple myeloma, and primary amyloidosis. Clin Cancer Res. 2002;8:2210-6.

13. Postema EJ, McEwan AJ, Riauka TA, Kumar P, Richmond DA, Abrams DN, et al. Initial results of hypoxia imaging using 1-alpha-D: -(5-deoxy-5-[18_{F]-fluoroarabinofuranosyl)-2-nitroimidazole (}

[18F]-FAZA). Eur J Nucl Med Mol Imaging. 2009;36:1565-73.

14. Bladé J, Samson D, Reece D, Apperley J, Björkstrand B, Gahrton G, et al. Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Myeloma Subcommittee of the EBMT. European group for Blood and marrow Transplant. Br j Haematol. 1998;102:1115-23.

15. Lokhorst HM, van der Holt B, Zweegman S, Vellenga E, Croockewit S, van Oers MH, et al. A randomized phase 3 study on the effect of thalidomide combined with adriamycin, dexamethasone, and high dose melphalan, followed by thalidomide maintenance in patients with multiple myeloma. Blood. 2010;115:1113-20.

16. Sonneveld P, Schmidt-Wolf I, van der Holt B, Jarari L, Bertsch U, Salwender H, et al. Bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase III HOVON -65/GMMG-HD4 trail. J Clin Oncol. 2012;30: 4513-6. 17. Hovenga S, Daenen SM, de Wolf JT, van Imhoff G, Kluin-Nelemans HC, et al. Combined Thalidomide

and cyclophosphamide treatment for refractory or relapsed multiple myeloma patients: a prospective phase II study. Ann Hematol. 2005;84:311-6.

(18)

4

18. Eckert AW, Lautner MH, Schütze A, Taubert H, Schubert J, Bilkenroth U. Coexpression of hypoxia-inducible factor-1α and glucose transporter-1 is associated with poor prognosis in oral squamous cell carcinoma patients. Histopathology. 2011;58:1136-47.

19. Weidenaar AC, ter Elst A, Koopman-Klein G, Rosati S, den Dunnen WF, Meeuwsen-de Boer T, et al. High acute myeloid leukemia derived VEGFA levels are associated with a specific vascular morphology in the leukemic bone marrow. Cell Oncol. 2011;34:289-96.

20. Boellaard R, O’Doherty MJ, Weber WA, Mottaghy FM, Lonsdale MN, Stroobants SG, et al. FDG PET and PET/CT: EANM procedure guidelines for tumour PET imaging: version 1.0. Eur J Nucl Med Mol Imaging. 2010;3:181-200.

21. Houwerzijl EJ, van den Heuvel FA, Blom NR, van der Want JJ, Mulder AB, Vellenga E. Sinusoidal endothelial cells are damaged and display enhanced autophagy in myelodysplastic syndromes. Br J Haematol. 2013;161:443-6.

22. Smith A, Wisloff F, Samson D, UK Myeloma Forum, Nordic Myeloma Study Group; British Committee for standards in Haematology. Guidelines on the diagnosis and management of multiple myeloma 2005. Br J Haematol. 2006;132:410-51.

23. van Lammeren - Venema D, Regelink JC, Riphagen II, Zweegman S, Hoekstra OS, Zijlstra JM. [18F]-Fluoro-dexoyglucose positron emission tomography in assessment of myeloma-related bone disease: a systemic review. Cancer. 2012;118: 1971-81.

24. Derlin T, Weber C, Habermann CR, Hermann J, Witsotzki C, Ayuk F, et al. (18)F-FDG PET/CT for detection and localization of residual or recurrent disease in patients with multiple myeloma after stem cell transplantation. Eur J Nucl Med Mol Imaging. 2011;38:493-500.

25. Van de Donk NW, Lokhorst HM, Dimopoulos M, Cavo M, Morgan G, Einsele H, et al. Treatment of relapsed and refractory multiple myeloma in the era of novel agents. Cancer Treat Rev. 2011;37:266-83.

26. Halmos GB, de Bruin L, Langendijk JA, van der Laan BF, Pruim J, Steenbakkers RJ. Head and neck tumor hypoxia imaging by [18F]-Fluoroazomycin-arabinoside ([18F]-FAZA)-PET. a review. Clin Nucl Med. 2014;39:44-8.

27. Bollineni VR, Kerner GS, Pruim J, Steenbakkers RJ, Wiegman EJ, Koole MJ, et al. PET imaging of tumor hypoxia using [18F]-Fluoroazomycin-arabinoside in stage III-IV non-small cell lung cancer patients. J Nucl Med. 2013;54:1175-80.

28. Martin SK, Diamond P, Gronthos S, Peet DJ, Zannettino AC. The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma. Leukemia. 2011;25:1533-42.

29. Fatrai S, Wierenga AT, Daenen SM, Vellenga E, Schuringa JJ. Identification of HIF-2alpha as an important STAT5 target gene in human hematopoietic stem cells. Blood. 2011;117:3320-30.